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目的探讨小鼠原始生殖细胞(primordial germ cells,PGCs)体外增殖及分化的条件。方法使用PGCs与睾丸支持细胞(SCs)共培养的方法进行PGCs体外增殖培养。用原代PGCs经悬浮培养获取类胚体(EBs),将EBs进行贴壁培养,观察其自然分化的过程。结果将PGCs与SCs共培养可获得大量PGCs集落,形似鸟巢状,组织化学与免疫组织化学染色显示提取的PGCs和增殖培养的PGCs集落的碱性磷酸酶(AKP)和阶段特异性胚胎抗原-1(SSEA-1)均呈阳性表达。使用悬浮培养的方法可获得EBs,将EBs贴壁培养后,可分化为上皮样细胞、成纤维样细胞和神经样细胞。结论小鼠PGCs与SCs共培养能在体外大量增殖,并保持其干细胞特性。悬浮培养可使PGCs形成EBs,将EBs贴壁培养可分化形成内、中和外三个胚层的细胞,即上皮样细胞、成纤维样细胞和神经样细胞。
Objective To investigate the conditions of in vitro proliferation and differentiation of mouse primordial germ cells (PGCs). Methods PGCs were cultured in vitro with co-culture of testicular cells (SCs). Embryoid bodies (EBs) were obtained by suspension culture of primary PGCs. EBs were cultured in adherent culture to observe their natural differentiation process. Results A large number of PGCs colonies were obtained after co-culture of PGCs with SCs. Nestin-like cells were observed. Histochemical and immunohistochemical staining showed that alkaline phosphatase (AKP) and stage-specific embryonic antigen-1 (SSEA-1) were positive. EBs can be obtained by suspension culture method. EBs can be differentiated into epithelial-like cells, fibroblast-like cells and neuron-like cells after adherent culture. Conclusion Co-culture of mouse PGCs and SCs can proliferate in vitro and maintain their stem cell characteristics. Suspension culture allows PGCs to form EBs, adherent EBs to cells that differentiate to form inner, middle and outer germ layers, ie, epithelioid cells, fibroblastoid cells, and neuronal cells.